Chromatographic purification of virus with brushite modified by autoclaving



United States Patent 3 478,145 CHROMATOGRAPHIC PURIFICATION OF VIRUS WITH BRUSHITE MODIFIED BY AUTOCLAVIN G Milton Lapidus, Rosemont, Pa., assignor to American Home Products Corporation, New York, N.Y., a corporation of Delaware No Drawing. Original application Sept. 15, 1964, Ser. No. 396,736, now Patent No. 3,368,867. Divided and this application Dec. 18, 1967, Ser. No. 691,184

Int. Cl. A61k 23/00 US. Cl. 42489 7 Claims ABSTRACT OF THE DISCLOSURE Influenza viruses, including B strains, are recovered from impure solutions by chromatographic adsorption on brushite which has been modified by autoclaving, followed by elution under controlled conditions of pH andmolarity. After desorption the purified viruses can be killed then formulated into vaccines useful to prevent influenza.

This application is a division of copending application Ser. No. 396,736, filed Sept. 15, 1964, and now US. 3,368,867.

This invention relates to the purification and concentration of virus. More particularly, this invention relates to a new adsorbent useful in the purification of influenza virus, to the method by which this new adsorbent is prepared and to the method by which the same is used in the production of purified and highly concentrated influenza virus.

BACKGROUND OF THE INVENTION In recent years increasing attention has been given to methods for the concentration and purification of various types of viruses including in particular the influenza virus. Earlier methods for such purification included the adsorption on, and elution from chicken red cells as proposed by Hirst, Science, 94, 22 (1941) and McClelland and Hare, Canad. Publ. Hlth. I. 32, 530 (1941). In 1943 Bodily et al. proposed the use of alum while Salk, Science, 101, 122 (1945) and Stanley, J. Exp. Med. 81, 193 (1945) proposed the use of calcium phosphate to precipitate and concentrate influenza virus. A short time later Taylor et al., J. Immunol, 50, 291 (1945) used a centrifuge technique to produce influenza virus on a commercial scale. It is with the latter two methods that the present invention is particularly concerned and directed to improving. According to the calcium phosphate v rus purification method which is more particularly described in the Journal of Gen. Microbiology 19, 451, (1958), Taverne et al., brushite (calcium phosphate (CaHPO,-2H O)) is described as a suitable adsorbent for the chromatographic purification and concentration of influenza virus. However, the brushite form when prepared according to the method described, is a very time consuming method and the adsorbent once so prepared is very unstable unless stored under very specific conditions. In addition the brushite form, as described in the Taverne article, while having been found reasonably effective in the purification of the A strain of influenza virus is substantially ineffective in the purification and concentration of the well recognized B strains of such virus. Consider now for a moment the widely used commercial centrifuge for producing purified influenza vaccine. According to this technique the virus, harvested in the form of infected allantoic fluid, for example, is delivered to a high speed centrifuge such as the Sharples centrifuge. During opera- 'ice tion of the centrifuge, the virus along with undesirable bacteria impurities, is plated out on the walls of the separator. Thereafter the virus is washed, scraped from the centrifuge walls, buffered and washed again in a blendor and once again centrifuged. The recovered virus is inactivated by the addition of Formalin and packaged in accordance with established standards utilizing the chick cell agglutination test as a measure of virus titer. The latter is determined in accordance with the National Institute of Health standards and is a measure of the viruses ability to agglutinate red blood cells. These tests units will be referred to hereinafter for convenience as CCA units. Though the centrifuge technique is used commercially, it is of very limited capacity in terms of throughput and provides a virus product which, while acceptable from a sterility point of view, does not in fact have the high degree of purity desired.

It is a primary object of the present invention therefore to provide a new chromatographic process for the concentration and purification of all strains of influenza virus.

It is a still further and additional object of the present invention to provide a process for preparing polyvalent influenza vaccine which includes both A and B strains.

These and other objects and advantages are attainable with the new adsorbent of the present invention, which is claimed in Ser. No. 396,736, now allowed, the method of its preparation and the methods in which the new adsorbent is used, the methods of use being the subject matter claimed in the instant application.

With respect to the composition aspects of the present invention, it has been found, quite unexpectedly, that if during the preparation of brushite, following the formation of the precipitated material, the same is subjected to autoclaving, an improved stable and effective adsorbent is obtained. Such an adsorbent properly activated, While being most eflfective in the purification of influenza virus generally, such as the A, C and D strains is quite unexpectedly effective in purifying B strains of influenza virus. The purification of the B strain as will be shown more clearly hereafter in the examples, has not previously been possible by a chromatographic process using the brushite adsorbent.

In addition to being able to purify the B strain of influenza virus, the use of autoclaved brushite, properly activated by a selective washing with distilled water to a pH of from about 6.8 to about 7.0, makes possible a very substantial increase in the purity of Sharples concentrated influenza virus. It has been found according to the method of the present invention that if Sharples concentrated influenza virus is adsorbed on autoclaved, activated brushite which has a pH of between 5.5 and 7.5 and preferably at about a pH of about 6.5 to 7.0, followed by an optional buffer wash and eluting the virus by treating with a buffer of increased molarity, a virus is eluted from the adsorbent which is from 5 to 10 times purer than is obtained by the conventional and standard Sharples centrifuge concentration technique.

It is an additional advantage of the present invention to utilize the new adsorbent thereof in the preparation of a purified and concentrated influenza virus without first concentrating the virus according to the Sharples centrifuge method. That is to say that the new brushite adsorbent of the present invention is so much more effective, for reasons which are not immediately clear, that a concentrated virus of a high degree of purity can be obtained at flow rates which obviate the necessity of first concentrating the virus according the Sharples methods now used commercially. Under these circumstances it is possible to obtain even the B strain of influenza virus at a purification factor of from 3 to 6 times greater and at a rate of recovery from 2 to 4 times greater than the rate at which the Sharples concentration method can effect separation of the desired influenza virus.

To prepare the new brushite (CaHPO -2H O) adsorbent composition for the present invention, substantially equimolar amounts of calcium chloride and sodium phosphate are delivered simultaneously to a common mixing vessel. After mixing has continued for a period of from /2 to 2 hours, preferably about 1 hour, supernatant liquid is removed from the mixing vessel by decantation. The brushite suspension is then washed with distilled water to bring the pH supernatant liquid to about 7.0. The brushite is autoclaved in a suitable apparatus for a period of from about to about minutes at a pressure of from about 10 to 30 p.s.i. which corresponds to a steam temperature of from about 115 C. to about 134 C. and allowed to cool to room temperature. Under these conditions the pH will normally be in the range of from about 5.0 to about 5.4. The modified brushite adsorbent is then washed with distilled water until a pH of about 6.5 is obtained. This activation is preferably though not necessarily carried out just prior to using the autoclaved brushite. The latter step, it has been found, is necessary to provide the adsorbent with the desired degree of activity. Quite unexpectedly at this pH the modified brushite is extremely effective in accomplishing the desired purification and concentration of the influenza virus in all strains, particularly in the B strain in either a concentrated form as by Sharples or in an unconcentrated form, such as for example from an allantoic fluid containing influenza virus.

The modified brushite adsorbent, prepared according to the method described, is more stable than forms previously available and is in addition able to concentrate B strains of influenza virus which previous brushite adsorbents were not able to accomplish.

DESCRIPTION OF THE INVENTION The instant invention contemplates in its broadest aspects a process for the purification of influenza virus which comprises:

(a) deaerating an autoclaved suspension of brushite;

(b) delivering the said suspension to a chromatographic column;

(c) washing said column with a buffer solution;

(d) flowing a virus-containing solution onto the column;

(e) washing said column with a buffer; and

(f) treating said column with a buffer for increased molarity.

According to the method of the present invention, the adsorbent in its highly activated form can be used to effect separation of all forms of the influenza virus. To accomplish this, the adsorbent will normally be deaerated and placed in a suitable extraction or chromatographic column to which the virus solution is delivered. The virus solution may take any form, including the concentrated form such as a Sharples concentrated form, as well as unconcentrated forms such as an infected allantoic fluid fresh from harvesting. Following deaeration, the suspension of autoclaved brushite is washed with 0.01 molar phosphate buffer solution to a pH of about 6.8. This is to condition the column to the adsorption of the virus. The latter has been similarly worked up to a pH of between 6.8 to 7.2 generally by means of dilute acid. The acid work up is particularly suitable when the virus is in the form of a Sharples concentrated influenza such as the B strain/ Great Lakes.

After being placed on the column, the virus adsorbs on the autoclaved brushite and impurities pass through in accordance with conventional chromatographic technique. Thereafter the column is washed with buffer and the virus eluted. The elution is accomplished by increasing the molarity of the bufler wash to about 0.5 molar at a pH preferably of about 6.8. It has been found that phosphate buffer is generally effective and preferred for elution. Normally, under the conditions described the virus solution will come off at a molarity of about 0.3 or 0.4

molar and is recognized by a turbidity in the take-01f vessel. The fraction containing the virus will also be highly opalescent.

The purified virus thus obtained may then be killed for example, by treatment with Formalinin the buffer solution in which it is contained. The killing is normally the next step in preparing a vaccine of the purified virus. If desired, the virus may be diluted in a buffer so as to be utilized in the preparation of the standard pool vaccine such as a standard 500 CCA/1 ml. vaccine. Such a pool would of course necessarily be required to meet standards established by public health authorities for influenza vaccines. The pool could normally include from 3 to 5 strains of purified killed virus properly balanced with respect to CCA units to provide the total pool with the 500 CCA/1 ml. content required. The latter technique is of course a standard and well known vaccine preparation procedure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The procedure which follows will provide a better understanding of the new virus purification adsorbent used in the present invention, and the method by which the same is prepared. The examples will describe the method by which improved purification of partially purified or concentrated influenza virus strains is carried out. They are illustrative, and are not to be construed to limit the scope of the invention in any manner whatsoever.

PROCEDURE Five moles of brushite is prepared from 10 liters of .5 M CaCl -2H O and 10 liters of .5 M Na HPO by metering both solutions at the rate of 4 liters per hour into a common mixing vessel. After delivery is completed, mixing is continued for one hour. After the brushite suspension settles, the supernatant is removed by decantation. Washing twice with 30 liters of distilled water brings the pH to 7.05. The brushite is then autoclaved for 15 minutes at 20 pounds steam pressure which corresponds to about 125 C. and allowed to cool to room temperature. The pH of the brushite modified by autoclaving is found to be 5.2. The brushite modified by autoclaving is washed with distilled water and with 0.01 molar buffer until pH 6.5 is reached.

The foregoing procedure sets forth the method by which the new and improved stable autoclaved brushite used in the present invention is obtained. In Example 1, which follows, there is provided a clear description of the method of using the new adsorbent to effect further purification of Sharples purified influenza B/GL.

EXAMPLE 1 The adsorbent brushite of the procedure is deaerated under vacuum and a column 4.4 x 9 cm. is poured and Washed with distilled water. Ten ml. of concentrated influenza B/GL, diluted 1:10, (0.01 M phosphate) adjusted to pH 7.0 with dilute HCl is passed into the column of adsorbent. The column is further washed with distilled water and eluted with .5 M, pH 6.8 phosphate buffer. Fraction 3 is highly opalescent and represents purified B/GL. (In the following tables, HA represents Chick Cell Hemagglutination units, also known as CCA units, a standard measure of virus titer.)

COLUMN CHROMATOGRAPHY OF SHARPLES PURIFIED INFLUENZA, B/GL Tot al Lowry ETA/mg.

Volume, Total, Protein, Lowry Purification Fraction m1. H mg. Protein Factor Original. 819, 200 25. 5 32, .5. 1 100 1, 600 7 2, 286

In a manner similar to Example 1 a comparative test was carried out to demonstrate the unexpected effect obta1ned by the use of the autoclaved brushite used in the present invention as compared to the non-autoclaved This pool met the standards of NIH for influenza virus brushite prepared according to prior technique. The matevaccine polyvalent, types A and B.

rial used in carrying out this comparative test, which was While the foregoing invention has been described with conducted substantially in the manner described in EX- sime degree of particularity, it is to be understood that ample 1, was infected allantoic fluid B strain/ Great Lakes the specific description is for purposes of illustration and influenza virus. is not intended in any way to act as a limitation on the EXAMPLE 2 Adsorbent Brushlte (prior art) Autoclave Brushite Conditions .001 M, pH 6.8 phosphate pH 8.0.

Virus. Inf. allantoic, B/GL Inf. allantoic, B/GL.

Put on Column, H 512,000 256,000.

Recovered, HA 51,200- 102,400.

Percent Recovery 40.

Purification Factor 0 2 It will be noted that the non-autoclaved brushite proscope of the invention. The latter is only to be limited by vided only a 10% recovery and no increase in purity as the claims appended hereto. compared to the autoclaved brushite which gave a yield of I claim: 40% and a purification factor of 2. In a similar manner a LA process for the purification of influenza virus, incomparative test was carried out between the autoclaved eluding B strains thereof, which comprises using an autoand non-autoclaved brushite with Sh-arples concentrated claved suspension of brushite prepared by mixing equi- B stram/ Great Lakes. molar amounts of EXAMPLE 3 h' z and Na HPO Autoclaved to form a brushite suspension, settling said suspensio 1- Adsorbent Brushite (prior art) Brushite lowmg t i perngtantdliquitli1 to separ r m ving h superna an lqlll an was ing the brushite sus ension 3333 9313323::::::"":: iaih slt??f iii EaifiQSB GL. with distilled water to a pH of about subjecting the Put on Column, HA 2 suspension to an autoclave pressure of from about 10 psi. to about psi. for a period of from about 10 minutes to about 30 minutes and washing the autoclaved suspen- The results above clearly demonstrate the Superiority 30 slon wlth suflicient distilled water so that the suspension Recovered, HA Percent Recoveryof the new adsorbent of the present invention over the has a P range of m about 5.5 to about 7.5 in the standard form of adsorbent. The anomalous value for re- Steps of;

covered HA in column 2 f Example 3 (the Virus (a) deaerat mg sa d autoclaved suspension of brushite;

covered appears greater than that added) is explained by dollverlng Sold Suspollsioh to a chromatographic the inherent error in the dilution method which sometimes makes it appear that the eluate is more concentrated in Washlug Sold column Wlth a Phosphate bufier Soluvirus. For the purpose of determining whether the coltlon ofhom obout p to about umn is effective to adsorb the virus, this error is ignored. flOWuIg a f on onto the column;

Only when a low titer is found on eluting the virus is the Washulg 831d Column With a Phosphate bufier luchange significantthis indicates that the column was tloh o P to about M r Water; and

not effective to hold the virus when the initial virus-conolutlhg the vll'us y treating Said column With a taining solution is passed through it. The dilution errors Phosphate buffer of from about M p to about in cases like those of Example 3 are treated by normalizing the recovery to 1 if is appears that a i titer 2. A process as defined in claim 1 which comprises:

is eluted than is introduced; the normalized value of 100% 5 o t u autoclaved brushite suspension;

is thus the next figure in the Said cohlmm (b) dellvermg said deaerated autoclaved brushite sus- As has been previously suggested, the product used in pollslou to a ohromatograoh ColuIuIl;

the present invention prepared according to the method Washlug Sold Column Wlth a Phosphate buffer SOhldescribed is ideally suited for use in the preparation of a tloh P to about to a P of about and vaccine which will meet present standards for pooled inh onto th column a Virus-Containing fluid,

fluenza virus polyvalent, types A and B. Example 4 which sold V1ru$"ohta1u111g fluld having a P of not more follows demonstrates this important aspect of the present than invention (e) washing said column with a phosphate buffer solu- EXAMPLE 4 tion of up to about 0.2 M to a pH of about 6.8; and

(f) eluting said virus with a phosphate buffer solution The A St a A1111 1/57u11d 1 of having a molarity of from about 0.3; up to about 0.5

influenza virus were purified on 3 molar columns of d H of about 5 g brushite prepared by the double drop technique. Allantoic 3 A processed as defined in claim 1 for the urification fluid (8.5-11.5 liters) was adjusted to pH 7.0, and .02 of a B Strain of influenza virus which comprisgs ph ph The lufluohza Vlrus on i 0 (a) deaerating under vacuum a suspension of said automately 9.5 x 30 cm. chromatographic columns of brushite claved brushite;

that were prewashed with .02 M, pH 7.0 phosphate buf- (b) introducing Said brushite into a chromatographic fer. Following the adsorption, the columns were washed column;

with .02 M, PH 7- Phosphate bufiel' and the was (0) washing said column with a phosphate buffer of eluted with .5 M, pH 7.0 phosphate bufler. The concenfrom about 0'01 M up to about Q02 M to a PH f tration of buffer was reduced by dilution of the purified about 68;

viru fractions and after formaledhyde inactivation and (d) introducing the B strain influenza-containing solumerthiolate addition, a pool of the four kinds of influenza tion diluted at about :1 and having a PH f 7 into virus strains was made, containing the following proporthe column;

tion of each strain: 70 (e) washing said column with distilled water; and

CCA llmts P 1111- (f) eluting said virus with a phosphate buffer having 4; 100 a pH of about 6.8 and a molarity of from about 0.3

Ann Arbor 1/57 100 M to about 0.5 M.

Jap 305 200 4. A process as defined in claim 1 for the purification Great Lakes 1739/54 1 0 of a B strain influenza virus which comprises (a) deaerating under vacuum said suspension of autoclaved brushite;

(b) introducing said brushite into a chromatographic column;

() washing said column with a phosphate buffer from about 0.01 M up to about 0.02 M to a pH of about 6.8;

(d) introducing the B strain influenza-containing solution diluted at 1:10 and having a pH of 7.0 onto the column;

(e) washing said column with distilled water; and

(f) eluting said virus with a phosphate bufler having a molarity of from about 0.3 M up to about 0.5 M and a pH of at least 6.8.

5. A process as defined in claim 1 which comprises:

(a) deaerating under a vacuum a suspension of said autoclaved, activated brushite;

(b) packing the deaerated autoclaved brushite in a chromatographic column;

(c) adjusting said column to a pH of about 6.8 by passing through it a phosphate butter solution of from about 0.01 M up to about 0.02 M;

(d) introducing into said column a virus-containing fluid having a pH of not more than about 8;

(e) washing said column with a phosphate buffer of up to about 0.02 M; and

(f) eluting said virus with a phosphate buffer having a molarity of from about 0.3 up to about 0.5.

6. A process as defined in claim wherein the eluted influenza virus in Step (f) is treated with Formalin in the buffer solution.

7. A process for the purification of influenza virus, including B strains thereof, which comprises using an autoclaved suspension of brushite prepared by mixing equimolar amount of CaCl -2H O and N32HPO to form a brushite suspension, settling said suspension allowing a supernatant liquid to separate, removing the supernatant liquid and washing the brushite suspension with distilled water to a pH of about 7, subjecting the suspension to an autoclave pressure of from about 10 psi. to about 30 psi. for a period of from about 10 minutes to about 30 minutes and washing the autoclaved suspension with suflicient distilled water so that the suspension has a pH range of from about 5.5 to about 7.5 in the steps of:

(a) deaerating said autoclaved brushite suspension under vacuum; (b) placing the deaerated autoclaved brushite suspension in a chromatographic column; (0) flowing onto the column a virus containing fluid; (d) washing the column with a phosphate buffer solution of up to about 0.02 M or water; and (e) eluting the virus by passing a phosphate buffer solution of from about 0.3 M up to about 0.5 M through the column.

References Cited UNITED STATES PATENTS 2,889,217 6/1959 LeBaron 23l09 X 3,197,374 7/1965 Hennessen et al. 3,386,882 6/1968 Eklund et al.

OTHER REFERENCES Taverne et al., J. Gen. Microbiol, vol. 19, pp. 451-461, December 1958.

RICHARD L. HUFF, Primary Examiner 

